Methods for producing highly sensitive potentiometric sensors

ABSTRACT

The invention relates to methods of preparation of highly sensitive potentiometric sensors with an electroconductive polymer film as a sensing element.

FIELD OF THE INVENTION

The invention relates to methods of preparation of highly sensitivepotentiometric sensors with an electroconductive polymer film as asensing element. The invention is applicable to the fields of medicine,biotechnology, agriculture, ecology as well as to environment monitoringand food quality assurance, particularly to laboratory testing ofbiological and environmental fluids performed for the purpose ofclinical diagnostics, proteomics, cell analysis, environmental andmanufacturing monitoring and research.

BACKGROUND TO THE INVENTION

The use of sensors with an electroconductive polymer film as a sensingelement is one of the most promising and attractive methods ofquantitative electrochemical analysis [1]. To date, a number ofelectrochemical sensors based on electroconductive polymers have beendescribed. They are distinguished by the principle of the measurement(amperometric, voltamperometric, chemoresistive, potentiometric) as wellas by the method of receiving the analytical signal (direct andnon-direct sensors).

The amperometric signal is received by applying to a sensor a constantvoltage from external source and measuring a level of current defined bychemical and/or biochemical reaction taking place within the sensor [2].Voltamperometric and chemoresistive sensors work similarly in principlebut with the difference that the applied voltage is not constant, beingchanged according to established parameters for a particular method [2,3].

As a rule amperometric and voltamperometric methods require expensiveequipment including an amperometer, external source of voltage orpotentiostat, a counter electrode and a reference electrode [2].

Potentiometric devices derive their responses from the change in redoxcomposition of the electroconductive polymer because of the chemicaland/or biological reaction, which accompanies the changes in the steadystate potential of the potentiometric sensor [2, 4, 5]. The authors ofthe present invention observe that potentiometric sensors have a numberof advantages over amperometric and voltamperometric sensors. One ofthem is that potentiometric methods do not require sophisticatedequipment. A potentiometric device usually comprises a sensor itself, areference electrode and a high impedance voltmeter [2, 6]. Secondly thesignal does not depend on a surface area or a shape of a sensor [2].Thirdly with a potentiometric method of measurement the problemsassociated with diffusion processes within the electrochemical cell andresulting in complicated constructions of electrodes (e.g. rotating discelectrodes) for amperometric and voltamperometric methods ofmeasurement, do not play a significant role [2, 4].

The use of potentiometric measurement can be as simple as pHmeasurement, and the potentiometric device can be similar to commercialpH or ion-selective electrodes.

All electrochemical sensors can be divided into two types, direct andindirect sensors.

A direct electrochemical sensor generates the signal immediately at themoment of interaction between an analyte and receptors immobilisedwithin or adsorbed onto the sensor. Examples of direct sensors areenzyme amperometric sensors [2, 7, 8], ion-selective potentiometricsensors [9, 10] and potentiometric immunosensors [11, 12, 13]. As a rulethe contact with an analyte and the measurement procedure are performedsimultaneously.

An indirect electrochemical sensor generates a signal due to thepresence of additional agents specific to an analyte carryingelectrochemically active labels. Examples of the sensors belonging toindirect group are amperometric and potentiometric enzyme sensors [2],potentiometric sensors sensitive to a change in surface potential [14,15] as well as voltamperometric and chemoresistive sensors [16, 17, 18].The contact between the sensor and an analyte and the measurementprocedure are separated in time and space.

The voltamperometric sensors can be described as intermediate betweendirect and indirect sensors. In this case there are no labelled agentsin the system but the incubation and measurement steps are separated intime and space and/or solute.

The most critical step in the manufacture of a highly sensitivepotentiometric sensor, having a conductive polymer layer as a sensingelement, is the formation of the polymer film on the conductive support.The support itself is usually a noble metal, carbon or semi-conductivematerial [19]. Electrochemical synthesis allows production of conductivepolymer films with defined chemical, electrochemical and mechanicalproperties [19].

The components of the polymerisation process include monomer(s), a polarsolvent and at least two electrodes (auxiliary and working) [19]. Asupporting electrolyte is usually included in the polymerisationsolution to increase conductivity of the solution and for doping thepolymer at the polymerisation step [19]. There are three main types ofelectrochemical synthesis: galvanostatic, potentiostatic andpotentiodynamic [19].

In the galvanostatic method a constant current from an external sourceis applied for period of time between working and auxiliary electrodesimmersed in the polymerisation solution. The reference electrode may beused to control the electrochemical potential of the working electrode[19, 20, 21].

In the potentiostatic method usually three electrodes are required. Thecurrent between the working and auxiliary electrodes is controlled by anamperometer set at a constant voltage from an external source (which isin its turn controlled by the reference electrode) applied between theworking electrode and auxiliary electrode for a certain period of time[19, 20, 21].

In the potentiodynamic method the voltage applied between the workingand auxiliary electrodes is not constant, but is changing according toestablished procedures [19, 20, 21].

The most important property for potentiometric enzyme- or immunosensorsis their redox sensitivity [4], because most of the enzyme reactions areredox processes accompanied by changing redox state of reactants. Theredox sensitivity of polymer-based sensors is completely defined byredox properties of the polymer film [22], which in their turn aredefined by the conditions and parameters of the polymerisation process.

A large number of publications are dedicated to research of redoxproperties of electroconductive polymers, e.g. polypyrrole [5, 22-27].In a number of these studies [22-23, 26] it was shown that two mainmechanisms are employed in the formation of the potentiometric signal.The irreversible change in the intrinsic redox state of theelectroconductive polymer is a consequence of the interaction betweenthe polymer layer and electrochemical active species and is referred toas a “corrosive type” of formation potentiometric response. This processis always accompanied by an ionic exchange between the polymer andsurrounding solution. Another mechanism is based on an electron exchangebetween the redox couples on the polymer surface via the polymer film.The intrinsic redox state of the polymer does not change in thisprocess, and an ionic exchange between polymer and solution does nottake place. In this case the electroconductive polymer behaves as ametallic potentiometric electrode and its behaviour can be described bythe Frumkin theory of electronic equilibrium [22, 28]. This is “metallictype” potentiometric response, and it is reversible. In reality bothmechanisms act simultaneously but one of them can be predominant [22-23,26, 27]. The “metallic type” is favoured for potentiometric redoxsensors because it provides the quicker and stronger response [26]. Itis possible to change the properties of the polymer film at thepolymerisation step making the “metallic” mechanism predominant [22-23,26].

Previously, the nature of a supporting electrolyte, and accordingly thenature of a dopant ion, were considered the only factors responsible formetallic properties of the polymer film [22-27, 29]. It was shown thatthe immobile anions embedded within the polymer film do not participatein ion exchange reactions, providing stability of intrinsic redox state[23, 26]. The examples of such electrolytes are dodecylsulphate [23],various dyes, e.g. indigo carmine and methylene blue [30, 31]. However,in all cited publications the concentration of dopant ions inpolymerisation solution is not considered as a factor responsible forimparting the metallic properties to the polymer.

The authors of the present invention consider the concentration of themonomer as well as the concentration of supporting electrolyte in thesolution for the electrochemical polymerisation as the most importantfactors for redox properties, and accordingly for redox sensitivity ofthe polymer. It is known that the concentration of the monomer can alsoinfluence the conductivity of polymer [19]. According to the data by theauthors of the present invention, the best redox sensitivity can bereached using the polymerisation solution with much lower concentrationsof monomer (<0.05M) than commonly used (0.05-0.5M). The authors of thepresent invention found that the ratio between the concentration of amonomer and supporting electrolyte is a key factor particularlyresponsible for redox properties and thickness of a polymer film. Theratio between monomer(s) and supporting electrolyte(s) as well as theirconcentrations had not previously been considered as factors responsiblefor redox properties of polymer film prior to the studies carried out bythe present inventors.

Despite the fact that there are a substantial number of methods forelectrochemical synthesis of the electroconductive polymer described inthe literature, none of them provide the conditions and parameters forproduction of highly sensitive sensors suitable for potentiometricdetection of biomolecules in low concentration.

Some examples of the prior art methods of electrochemical synthesis aregiven below.

Potentiostatic methods for preparation of electrochemicalpolypyrrole-based sensors from aqueous solutions in the presence of asupporting electrolyte are described in [39, 42, 43, 44 and 45].

Taniguchi et al [33] described the method for growing polypyrrole filmfrom organic solvents in the presence of a supporting electrolyte usinggalvanostatic regime. The generated polymer films were used for thepreparation of a direct potentiometric immunosensor. The maindisadvantage of this method is that the sensitivity of the sensorsproduced by this method is poor (mg/ml of IgG). Another disadvantage isthat the organic solvents used in such a method are highly toxic.

Other galvanostatic methods where water is used as a solvent aredescribed in [13, 16, 17, 34, 35, 36, 37, 38, 39, 40, 41].

A potentiodynamic method of electrochemical polymerisation for synthesisof electroconductive polymers from aqueous solutions in the presence ofa supporting electrolyte for preparation of electrochemical sensors hasbeen described [9, 10, 27, 46, 47, 48, 49].

Most of the sensors produced by the methods described in the articlesmentioned above were not intended to use for potentiometric measurement,but for other types of electrochemical measurement or entrapment. All ofthem are unsuitable for potentiometric assays requiring high analyticalsensitivity, precision and stability. The methods cannot be developeddirectly to the method described in the present invention to provide therequired properties of the sensors. The measuring procedures are alwaysmore complicated and takes longer than the one which is described inthis invention.

The authors of the present invention have also described thepotentiodynamic method of preparations of potentiometricpolypyrrole-based sensors [50, 51].

Despite the possibility to use polymerisation solutions with lowconcentrations of monomer(s) in the potentiodynamic and galvanostaticregimes [19], most of the cited publications describe use ofconcentrations of 0.05M and higher. As it has been stressed by theauthors of the present invention, polymer films grown from concentratedmonomer solutions do not have high redox sensitivity and thereforecannot be used as a highly sensitive element of the polymer-basedpotentiometric sensor.

Low concentrations of monomer were mentioned in only one single study[48], but the authors used high concentrations of the supportingelectrolytes (0.5M) and supporting electrolytes with highly mobileanions (KCl, KNO₃, NaClO₄, Na₂HPO₄), which are actively interactive inionic exchange between polymer and surrounding solution. Thepotentiometric response of such sensors belongs mainly to thecorrosion-type mechanism. This type of sensor is not sensitive enoughfor measurement of biological redox reactions, where clinically orenvironmentally relevant concentrations of analyte may routinely occurin the range of nanomoles, femtomoles or attomoles. Other studies alsoused highly mobile anions as dopant ions, resulting in sensors, whichexhibit low sensitivity. The work of Hulanicki et al [27], where theauthors doped polymer with Cl⁻, can be given as an example. In this casethe sensors demonstrated redox sensitivity only in presence of very highconcentrations of redox couples (about 0.5M).

Other studies [9, 49, 51] used a suitable dopant ion-sodiumdodecylsulphate, but high concentrations of polymer (0.05-0.3M), whichis again not suitable for preparation of highly sensitive potentiometricredox sensors.

The present inventors have defined the main factors, which, incombination, are responsible for the redox properties of the polymerfilm and as a consequence are able to produce polymer-basedpotentiometric sensors with higher sensitivity than those described inthe prior art. These factors are the concentration of a monomer(s);nature and concentration of the supporting electrolyte; parameters ofthe polymerisation process; ratio between the concentrations of monomerand supporting electrolyte. The prior studies relate to only one or twoparameters or conditions and not their synergistic influences orinterferences. The authors of the present invention have found that ahighly sensitive polymer film can be produced by combining all of thefactors mentioned above.

SUMMARY OF THE INVENTION

The present invention relates to the production of highly sensitivereproducible and long-term stable polymer-based potentiometric sensors.

In a first aspect, the invention relates to a method of electrochemicalsynthesis of a polymer film with high redox sensitivity, which can serveas a sensing element of highly sensitive potentiometric chemical,enzyme- and immunosensors. There are three main types of electrochemicalsynthesis: galvanostatic, potentiostatic and potentiodynamic [19]. Allof these can be used either alone or in combination to electrochemicallygrow the polypyrrole layer.

Thus, the invention provides a method for producing highly sensitivepotentiometric sensors by coating of electrically conductive electrodeswith an electroconductive polymer, which method comprises the steps of:

(a) preparing an aqueous solution for electrochemical polymerisationcomprising monomeric units of the electroconductive polymer at aconcentration in the range of from 0.002 to 0.05M; and a supportingelectrolyte, which also serves as a doping agent, at a concentration inthe range of from 0.0001 to 0.005M;(b) assembling an electrochemical polymerisation cell comprising thesolution for electrochemical polymerisation, an auxiliary electrode, oneor more working electrodes to be coated with electroconductive polymer,and optionally a reference electrode; and(c) coating the working electrode(s) with a polymer film by theelectrochemical synthesis of polymer from the electrochemicalpolymerisation solution using at least one of the followingelectrochemical regimes:(i) applying a cyclic voltage in the range of from −0.2 to +2.0 V vsAg/AgCl reference electrode between the working electrode(s) to becoated and the auxiliary electrode;(ii) applying a constant current in the single or multiple current stepswith given current density in a range of from 0.01 to 1 mA/cm² betweenworking electrode(s) to be coated and auxiliary electrode for definedperiod of time such that final quantity of electricity passed throughworking electrode(s) will lie in a range of from 10 to 250 mC/cm²;(iii) applying a constant potential in a single or multiple potentialsteps at the range of from 0 to 3 V between working electrode(s) to becoated and a reference electrode for defined period of time such thatfinal quantity of electricity passed through the working electrode(s)will lie in a range of from 10 to 250 mC/cm²;(iv) any other electrochemical regime, where all the solutionconcentrations and electrochemical parameters previously stated areadhered to.

The basis of this method is the research conducted by authors, fromwhich the following conclusions can be drawn:

-   -   Redox sensitivity of polymer (e.g. polypyrrole)-based        potentiometric sensors increases significantly (sharply), when        the electrochemical synthesis is performed from the solutions        with low concentration of monomers (e.g. pyrrole) (<0.05M).    -   The increase in redox sensitivity is observed for a range of        monomer (e.g. pyrrole) concentrations in the range from        0.002-0.05M in the presence of a supporting electrolyte, which        serves as a doping agent, for example sodium dodecylsulphate.    -   The ultimate increase of redox sensitivity is observed when the        ratio between the molar concentrations of monomer (e.g. pyrrole)        and supporting electrolyte is approximately 25:1 (although other        ratios may be used within the scope of the invention) and either        one or more of the following electrochemical polymerisation        methods are used:    -   i) Potentiodynamic Regime: A cyclic voltage in the range        −0.2-+2.0 V (vs Ag/AgCl reference electrode) is applied between        the working electrode(s) (to be coated) and the reference        electrode.    -   ii) Galvanostatic Regime: One or more (cascade) levels of        current steps are applied in which the total charge passed        during polymerisation is in the range from 10-250 mC/cm²    -   iii) Potentiostatic Regime: One or more (cascade) levels of        potential steps are applied between the working electrode and        the reference electrode in which a total charge passed during        polymerisation is in the range from 10-250 mC/cm².    -   The use of more than one level of current in galvanostatic        regime and/or more than one level of applied potential in        potentiostatic regime allows tight control of the properties of        the sensor, and therefore production of sensors with better        performance characteristics, e.g. more sensitive, than protocols        using a single level of current or potential during        electropolymerisation.    -   The concentrations of the monomer(s) and the supporting        electrolyte(s), the ratio between them, and the applied        polymerisation procedure synergistically influence redox        sensitivity of polymer (e.g. polypyrrole)-based sensors.

The inventors' observations are unexpected because, as mentioned above,the relationship between redox sensitivity and such parameters asmonomer(s) concentration, nature and concentration of supportingelectrolyte and the ratio between them and parameters of thepolymerisation procedure were not considered in previous publications.There is a strong correlation between redox sensitivity of the polymerfilm and the final analytical sensitivity of the sensor. A veryimportant aspect of the present invention is that the observation thatit is possible to regulate redox sensitivity by changing the compositionof solution and parameters of the polymerisation process. It is possibleto produce sensors for determination of some viral infections (e.g. HIV,HBsAg), where sensitivity at the level of femtomoles is required.Sometimes the range of interest for an analyte lies within higherconcentrations, e.g. Digoxin (0.5-5 ng/ml) or IgE (20-1000 ng/ml).

The measuring range can be shifted to higher concentrations or extendedby changing the set of parameters for the electrochemical polymerisationprocess or/and composition of the substrate system for furthermeasurement procedure.

To summarise: analytical sensitivity and measuring range can be tailoredfor a particular analyte. This is achieved by the unique combination ofthe defined concentrations of monomers(s), supporting electrolyte inpolymerisation solution and defined polymerisation regime in conjunctionwith the following treatment of the sensors.

This invention provides potentiodynamic, galvanostatic andpotentiostatic methods for producing highly sensitive polymer-coated(e.g. polypyrrole-coated) potentiometric sensors by electrochemicalpolymerisation of monomers (e.g. pyrrole). Any method can be used incombination with other methods.

The parameters for potentiostatic regime were derived (calculated) fromgalvanostatic procedures and tested in experiment. For the same growthsolution and design of sensors, the potential and currents are dependenton each other. The potential recorded at the certain applied current canbe applied in potentiostatic regime would give approximately the samecurrent as in galvanostatic procedure.

The exact values in each polymerisation method can slightly varydepending on the properties of the conductive or semi-conductive layer,but in general all procedures give comparative results and can besuccessfully applied to any type of electroconductive orsemi-electroconductive support.

Parameters for the polymerisation process can be calculated using eitherthe geometric surface area or the electrochemical surface area of thesensors onto which the polymer is to be deposited.

Various sensor designs have been tested by the inventors. A preferreddesign of sensor consists of a screen printed circular electrode with adiameter of 1.5 mm² giving a geometric area of 1.77 mm². Other designsmay be envisaged and the invention is not to be construed as limited tothis particular design. The method used for the calculation of theelectrochemical surface area was by placing the electrode in a solutionwith a redox species (e.g. 5 mM ferrocyanide) and a supportingelectrolyte (e.g. 0.1 M NaNO₃). The potential of the electrode isstepped from a potential where no current flows to a potential where allthe reduced species is oxidised and the resulting current is recordedwith time (chronoamperometry). The shape of the current response withtime is given by the Cottrell equation:i=(nFACD^(0.5))/(π^(0.5)t^(0.5))Where n=number of electrons transferred=1, F=Faraday (96480 C mol),A=surface area of electrode, D=diffusion coefficient of reduced species,C=concentration of reduced species, i=current, t=time. If the current isplotted against t^(−0.5) then the data should be linear and the area canbe calculated from the slope. Alternative methods of estimating theelectrochemcial surface area can also be used.

A preferred potentiodynamic method comprises the steps of:

-   -   a) preparing an aqueous solution for electrochemical        polymerisation comprising monomers (e.g. pyrrole) at a        concentration in the range of 0.002-0.05M, and a supporting        electrolyte, which also serves as a doping agent, at a        concentration in the range of 0.00.01-0.005M    -   b) assembling an electrochemical polymerisation cell comprising        the solution for electrochemical polymerisation, an auxiliary        electrode, a reference electrode and one or more electrodes to        be coated with a polymer film, wherein the electrodes to be        coated comprise a conducting or semi-conducting layer;    -   c) applying a cyclic voltage in the range −0.2-15+2.0 V (vs        Ag/AgCl reference electrode) between the electrode(s) to be        coated and the reference electrode to coat the electrode(s) with        a polymer film by the electrochemical synthesis of polymer from        the electrochemical polymerisation solution.

A preferred potentiostatic method comprises the steps of:

-   -   a) preparing an aqueous solution for electrochemical        polymerisation comprising monomers (e.g pyrrole) at a        concentration in the range of 0.002-0.05M, and a supporting        electrolyte, which also serves as a doping agent, at a        concentration in the range of 0.0001-0.005M    -   b) assembling an electrochemical polymerisation cell comprising        the solution for electrochemical polymerisation, an auxiliary        electrode, a reference electrode and one or more electrodes to        be coated with a polymer film, wherein the electrodes to be        coated comprise a conducting or semi-conducting layer;    -   c) applying a constant potential in a single or multiple        potential steps at the range 0-3 V between working electrode(s)        to be coated and reference electrode for defined period of time        such that final quantity of electricity passed through the        working electrode(s) will lie in a range 10-250 mC/cm²;

This invention also includes a galvanostatic regime of electrochemicalsynthesis as an alternative to the potentiodynamic one. In this case thequantity of electricity passed through working electrode(s) to be coatedlying within range 10-250 mC/cm² (preferably 10-60 mC/cm²) is a resultof applying a constant current with given current density betweenworking electrode(s) and auxiliary electrode for defined period(s) oftime. The single or multiple current steps can be used. The currentdensity can be varied within the range 0.01-1 mA/cm².

The present invention provides a galvanostatic method for producinghighly sensitive polymer-coated potentiometric sensors byelectrochemical polymerisation, which comprises the steps of:

-   -   a) preparing an aqueous solution for electrochemical        polymerisation comprising monomers (e.g. pyrrole) at a        concentration in the range of 0.002-0.05M; and a supporting        electrolyte, which also serves as a doping agent, at a        concentration in the range of 0.0001-0.005M;    -   b) assembling an electrochemical polymerisation cell comprising        the solution for electrochemical polymerisation, an auxiliary        electrode and one or more electrodes to be coated with a polymer        film, wherein the electrodes to be coated comprise a conducting        or semi-conducting layer;    -   c) applying a constant current in the single or multiple current        steps with given current density in a range 0.01-1 mA/cm²        between working electrode(s) to be coated and auxiliary        electrode for defined period of time such that final quantity of        electricity passed through working electrode(s) will lie in the        range 10-250 mC/cm²(preferably 10-90 mC/cm²).

The reference electrode can be used to monitor the galvanostatic processor alternatively a two electrode system may be employed in which aseparate reference electrode is not used. In addition, combinations ofthe different regimes can be used in one polymerisation process. Forexample, in one embodiment firstly the working electrode(s) can becoated with polymer film in galvanostatic regime, then the additionallayer of polymer can be applied using the potentiodynamic orpotentiostatic regime. The opposite is also possible. It is alsopossible to combine two or more galvanostatic regimes. Thesecombinations give more flexibility in controlling redox condition of thepolymer film.

The use of multiple currents applied for different times ingalvanostatic regime allows tailoring redox properties of the polymerfilm.

The combination of potentiodynamic cycle(s) or potentiostatic step(s)and galvanostatic step(s) can be used to tailor the redox properties ofthe polymer film.

The possibility to tailor redox properties by combining more than onepolymerisation regime and use of multiple successive currents ingalvanostatic regime for preparation of biosensors has not beenpreviously described and is a novel part of this invention.

The method of the invention may be used for applying a polymer film ontoa single electrode or a number of electrodes (greater than one) in onestep. The ability to coat multiple electrodes in a single polymerisationreaction increases reproducibility and decreases the cost of themanufacturing process. In contrast to previous works, where only asingle electrode was coated, the authors of the present inventionconnect a number of conductive or semi-conductive electrodes to becoated in one block with one single electrical contact, in anelectrochemical polymerisation cell, comprising an auxiliary electrodeand-for potentiodynamic and potentiostatic regimes-the referenceelectrode and the solution for electrochemical polymerisation. Allelectrodes to be coated behave as one single working electrode.Theoretically the number of electrodes to be coated is not limited andcan reach tens or even hundreds at a time.

The inventors have further observed that highly sensitive polymer-coatedsensors can be produced with the use of combinations of electrochemicalpolymerisation regimes and/or the use of multiple current or potentialsteps in the polymerisation regime, without limitation to the use of low(<0.05 M) concentrations of monomer in the electrochemicalpolymerisation solution.

Therefore, the invention also relates to multi-step methods forelectrochemical polymerisation using combinations of electrochemicalregimes and/or electrochemical regimes with multiple current orpotential steps.

In particular, the invention relates to the following:

A method for producing highly sensitive potentiometric sensors bycoating of electrically conductive electrodes with an electroconductivepolymer, where two or more current steps are applied in a galvanostaticregime.

A method for producing highly sensitive potentiometric sensors bycoating of electrically conductive electrodes with an electroconductivepolymer, where two or more potential steps are applied in apotentiostatic regime.

A method for producing highly sensitive potentiometric sensors bycoating of electrically conductive electrodes with an electroconductivepolymer, where two or more polymerisation regimes, preferably selectedfrom galvanostatic, potentiodynamic, potentiostatic, or otherelectrochemical regimes, are applied.

Preferred galvanostatic, potentiodynamic, potentiostatic regimes arethose described above in connection with the first aspect of theinvention.

As aforesaid, in these methods no limitation is placed on theconcentration of monomers or background electrolyte used in theelectrochemical polymerisation solution. Thus, the methods can be usedwith the higher concentrations of monomers described in WO 00/11473.

The scope of the invention extends to polymer-coated potentiometricsensors produced according to the methods of the invention, and also touse of these sensors. In particular, the potentiometric sensors may beused in methods for electrochemical detection of analytes (such as thosedescribed in WO 00/11473, WO 96/02001, etc.), also in methods forpotentiometric detection of enzymatic activity (e.g. methods formeasuring the activity of enzymes positioned proximal to the sensorsurface) and in methods for potentiometric analysis of whole cells (forexample using the techniques described in the applicant's co-pendingapplication GB 0207116.5).

In a further aspect the invention also provides a method of treatment ofelectroconductive polymer-coated sensors after the deposition of thepolymer film to increase both long-term stability and analyticalsensitivity of sensors. This treatment may be applied to sensorsprepared according to the method of the invention, described above, butmay also be applied to polymer-coated potentiometric sensors preparedaccording to prior art methods of electrochemical polymerisation (see WO96/02001, WO 0.00/11473, etc).

The treatment method comprises two stages. The first stage is a thoroughwash of the polymer-coated sensors by de-ionised water after theelectrochemical polymerisation. At this stage non-embedded dopant anionand traces of monomer(s) are eliminated from the polymer film. Removalof a monomer(s) is necessary, because monomers can be oxidised duringthe storage changing the intrinsic redox properties of the polymer film.Removal of non-embedded dopant ion is also necessary, because the mobilecounterion can compromise the metallic properties of the polymer [22,23] decreasing redox sensitivity. Particularly, an excess of sodiumdodecylsulphate denatures protein molecules [16] and decreasesadsorption of biomolecules. That could be a negative point for furtherimmobilisation of biomolecules within or onto a polymer film.

The second stabilising step is a removal of unbound water from thepolymer film. It is very important, because interactions of unboundwater with the polymer change the intrinsic redox properties of thepolymer film over a period of time. This is why there are manypublications regarding the instability of polymer (e.g. polypyrrole)films over a period of time because the most popular storage method(i.e., in wet-format) [13, 16, 32, 35, 41] leads to instability of theelectrochemical characteristics and non-reproducibility of the resultsof the potentiometric (or other electrochemical) measurements.

These two treatment steps allow sensors to be stored for a long timewithout changes of their working characteristics, which is veryimportant for the commercial manufacture of sensors.

Another reason why the polymer-based sensors produced according to themethod described in the present invention are stable for a long periodof storage, is because the polymer films are much thinner than commonlyused (“translucent” films). In such thin films the post-polymerisationprocesses end very quickly and do not further affect the polymerproperties.

A further aspect of the invention relates to a method for testing thesensitivity of the polymer-based sensors produced by the methoddescribed above. This test can show up very small differences in sensorperformance and enhance the ability to tailor the sensors properties.These differences could not be differentiated by generally appliedelectrochemical procedures, for example measuring the potential bymeasuring the open circuit potential of the sensors in an electrolytesolution.

It is necessary to have a universal, reproducible and quick test forsensitivity of the obtained sensors in order to evaluate reliably therelationship between conditions of the preparation of the sensors(polymerisation procedure and following treatment) and their analyticalsensitivity. This test can also be used as a quality control for alltypes of sensors and for the future sensor manufacturing process.

The authors of the present invention have found that the reactionbetween immobilised streptavidin and biotin-labelled horseradishperoxidase (HRP) is a simple, quick and reliable test suitable fortesting the analytical sensitivity of the sensors. All components forthis test are commercially available and certified.

Therefore, the invention provides a method for testing the analyticalsensitivity of a polymer-coated potentiometric sensor, which methodcomprises the following consecutive steps:

(a) coating the sensor with streptavidin by passive adsorption;

(b) applying a sucrose protective film to the streptavidin coatedsensor;

(c) bringing the sensor obtained in step (b) into contact with asolution containing a known concentration of biotin-labelled horseradishperoxidase for a defined period of time;

(d) monitoring the electric potential difference between the sensor anda reference electrode when both are immersed in a basic electrolytesolution;

(e) replacing the basic electrolyte solution with an enhancerelectrolyte solution having identical composition to the basicelectrolyte solution except that it additionally contains a substratefor horseradish peroxidase and monitoring the electric potentialdifference between the sensor and reference electrodes when immersed inthe enhancer electrolyte solution;(f) calculating the difference between the electric potential differencemeasurements obtained in steps (d) and (e) and comparing the resultobtained with reference results obtained with use of a pre-definedstandard sensor or other sensors evaluated at the same time.

Steps a) and b) can be combined in one step: a drop of Streptavidin insucrose solution can be placed onto the sensor and dried. Any additionalcomponents, e.g. blocking components or stabilisers, can be added to thesolution. The washing step may be required before performing the assay.

In a preferred embodiment the basic electrolyte solution used in step(d) may comprise an H-donor.

The above method provides a rapid, standardised potentiometric assaywhich may be used to quickly evaluate the analytical sensitivity of agiven potentiometric sensor and to determine the effect of, for example,changes in the composition of the electrochemical polymerisationsolution on the final analytical sensitivity of the sensor. Analyticalsensitivity is preferably evaluated relative to a standard or referencesensor, which is selected by the user. The standard or reference sensoris chosen merely to provide a basis line (or reference line) againstwhich other sensors may be compared. The precise characteristics of thereference sensor are not material to the invention.

It is important that assay parameters, i.e. concentrations of reagents,incubation times etc are standardised in order to allow meaningfulcomparison between results obtained with different sensors. However, theprecise values of these parameters are not material and may be selectedby the user. The skilled reader will appreciate that suitable assayparameters may be determined by routine experiment. One example of amodel assay is given in the accompanying examples.

The standardised assay is a development of the method described in theapplicants' International application WO 00/11473 and steps (a) and (c)to (e) may be performed as described in WO 00/11473, the contents ofwhich are incorporated herein by reference.

Step (b) results in coating of the sensor with a protective layer ofsucrose. This step is important as the sucrose layer prevents loss ofactivity of the adsorbed streptavidin and also helps to prevent oxygenand moisture access to the polymer layer. As illustrated below, thesucrose coating is conveniently applied by dipping the sensor into asucrose solution (typically 1-25%, most preferably 10% sucrose) orapplying a drop of sucrose solution containing streptavidin, if thesteps (a) and (b) are combined, then drying the sensor (preferably at30-40° C. for approximately 8-12 hours).

The utility of applying a coating of sucrose to the sensor is notlimited to the standard model assay system. Rather, any potentiometricsensor comprising a conductive electrode coated with a layer ofconductive polymer, particularly polypyrrole, can be coated with aprotective layer of sucrose. Furthermore, other protective substancescan be used instead of sucrose with equivalent effect.

Therefore, in a further aspect the invention provides a potentiometricsensor comprising an electroconductive electrode coated with anelectroconductive polymer, characterised in that a coating of aprotective substance is applied on top of the electroconductive polymer.

Suitable protective substances are those that act as proteinstabilisers. Suitable examples include, inter alia, trehalose, inositol,cellobiose and lactitol, as well as sucrose. It is also possible to usemixtures of these substances with polymers such as dextrans orpolyglycols. The protective coating is generally applied by immersingthe sensor in a solution of the protective substance or by any othersuitable method, e.g. placing a drop of protective solution orscreen-printing (e.g. a 1-25% solution of trehalose, inositol,cellobiose, lactitol or sucrose) and then drying the sensor. Any othersubstances to be applied to the sensors, such as bioreceptors, e.g.streptavidin, antibodies, peptides, etc., as well as blocking agents,stabilisers, etc. can be added to the protective solution and applied atthis step.

In a further aspect the present invention provides a potentiometricsensor comprising an electroconductive polymer, characterised in that itcan be used in any analysis with potentiometric detection step, e.g.enzymatic assays or cell analysis, where the measurable change inpotential of the polymer layer due to redox, pH, or ionic changes due toenzymatic activity or cell metabolic activity occurs. For example, cellscan be attached to the surface by growing there directly or via affinityinteractions and the change in potential can be detected bypotentiometric measurement described in the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the relationship between the potentiometric responsefrom a polypyrrole potentiometric sensor coated with streptavidin andthe concentration of biotinylated horseradish peroxidase (Bt-HRP) forpotentiometric sensors coated with polypyrrole films, grown frompolymerisation solutions containing different concentrations of pyrrole.

FIG. 2 illustrates the relationship between the potentiometric responsefrom a polypyrrole potentiometric sensor coated with streptavidin andthe concentration of horseradish peroxidase (HRP) for potentiometricsensors coated with polypyrrole films, grown from polymerisationsolutions containing different concentrations of SDS.

FIG. 3 illustrates the effect of variation in upper boundary potentialon the analytical response and the shape of the curve (signal vs HRPconcentration) for a polypyrrole-coated potentiometric sensor.

FIG. 4 illustrates the effect of variation in quantity of electricitypassed through the working electrode on the analytical response and theshape of the calibration curve for a polypyrrole-coated potentiometricsensor.

FIG. 5 illustrates the effect of various galvanostatic polypyrrolegrowth regimes on the response to biotinylated HRP (Bt-HRP). Each growthregime consists of a sequence of galvanostatically controlled currentsteps in which each step may be applied for a time between 10 to 1000 s.

FIG. 6 illustrates the effect of the electrode design on the response tobiotinylated HRP (Bt-HRP). The “linear” electrode design consists of anelectrode design in which the width of the electrode is significantlyless than the length of the electrode. The “circular” design consists ofa circular or disc shaped electrode.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention is used for the production of highlysensitive potentiometric sensors by coating of electrically conductiveelectrodes with an electroconductive polymer.

The electrically conductive electrode to be coated withelectroconductive polymer may be essentially any suitable electrodecomprising a conductive or semi-conductive layer. Suitable electrodesinclude standard potentiometric electrodes possessing metallic orquasi-metallic conductivity which are stable in aqueous media. Theelectrode preferably consists of a plastic support with an adhesivelayer (carbon or copper) with a conductive substrate (preferably gold)electrochemically plated or directly screen-printed onto the plasticsupport. The reference electrode, e.g. Ag/AgCl reference electrode,which is required for potentiometric detection step can be placed on thesame support as the sensing electrode by any method, for examplescreen-printed. An external commercial reference electrode can be usedas well.

Any lay outs of a final sensor product are possible, for example,“dip-stick”, multiwell plates containing integrated electrochemicalsensors for use in methods of electrochemical analysis.

The aqueous electropolymerization solution typically comprises monomericunits of the electroconductive polymer at a concentration in the rangeof from 0.002 M-0.05 M, preferably 0.002 M-0.02M, more preferably 0.0025M-0.15 M, more preferably 0.005 M-0.01 M in distilled water (e.g.MilliQ) and a supporting electrolyte at a concentration in the rangefrom 0.0001 M-0.005 M, preferably, 0.0001 M-0.002, preferably 0.0001M-0.0015 M, more preferably 0.0001-0.001 M. Other polar solvents may besubstituted for distilled water.

Suitable monomers include pyrrole, furan, thiophene or other, withpyrrole being most preferred. Combinations of two or more of thesemonomers may also be used, leading to the production of conductivecopolymers.

The preferred supporting electrolyte is sodium dodecylsulphate but otherelectrolytes, the anions of which are immobile within the polymer films,may be used. The electrolyte also serves as a doping agent.

Most preferably the electrochemical polymerisation solution consists ofan aqueous solution of monomers and supporting electrolyte. However, itis to be understood that other components may be added to thepolymerisation solution such as, for example, components which providespecific functional groups which can be used as linkers for bioreceptorsor for chemical modification of the sensor surface (see WO 00/11473).

The ratio between the concentrations of monomers and supportingelectrolyte in the polymerisation solution is preferably in the rangefrom 2:1 to 30:1, and more preferably in the range from 5:1 to 30:1. Aratio of approximately 25:1 is the most preferred.

The most preferred compositions, though not limiting to the overallscope of the invention, are 0.005-0.01 M monomers (pyrrole being mostpreferred) with 0.0002 M electrolyte (SDS being most preferred) or0.0075-0.01 M monomers (pyrrole most preferred) with 0.0017 Melectrolyte (SDS most preferred).

The electrochemical polymerisation is carried out in a two- orthree-electrode system comprising of electrode(s) to be coated (alsoreferred to herein as the “working electrode”), the auxiliary electrodeand the reference electrode. In the case of the two electrode system(galvanostatic regime) the reference electrode would not be used.Suitable assemblies have been described in the prior art (see WO00/11473 and references contained therein). Multiple working electrodescan be combined in a block with one electrical contact.

The auxiliary electrode is preferably made of platinum, other noblemetal or other inert conductive material such as graphite or carbon. Theauxiliary electrode should have a surface area greater than total areaof all working electrodes [53]. In order to decrease the uncompensatedsolution resistance in the polymerisation solution, the referenceelectrode should be positioned as close as possible to the workingelectrodes. [20, 21, 53]. A constant distance between the workingelectrodes and the reference electrode is preferable. The conventionalAg/AgCl or calomel electrode can serve as a reference electrode.

A potentiostat may be used for performing the electrochemical synthesis.In case of potentiodynamic regime a cyclic voltage within the range of−0.2-+2.0 V (vs Ag/AgCl reference electrode) at a scan rate ofpreferably 50-100 mV/s for preferably 1-15 cycles is applied between thereference electrode and the working electrode(s) to be coated. Thecurrent is recorded at the auxiliary electrode.

The shape of the voltammetric curve and total quantity of electricitypassed through the working electrode(s) are controlled parameters forpolymer formation. The quantity of the electricity passed during eachcycle must not differ more than 5% from the first cycle.

In case of the galvanostatic regime one or more constant current stepscan be applied between the working electrode(s) to be coated and theauxiliary electrode within the range of 0.01-1 mA/cm² for time of100-1000 s. The number of applied current steps is not limited. One tofive steps have been used by authors so far. The preferred totalduration of polymerisation is 150-600 s.

The galvanostatic regime is more preferable. It is less expensive andeasier to control than other regimes, and the equipment is lesssophisticated. The use of different applied currents allows tailoring ofthe redox properties of the polymer films with a very high precision.The use of the galvanostatic regime also allows precise control of anoxidation level of the resulting polymer film.

In some particular cases the sequential use of galvanostatic andpotentiodynamic or potentiostatic (and vice versa) regimes in onepolymerisation process is possible. For example the main polymer film isformed at the low potential using small constant current ingalvanostatic regime and after that the additional amount of polymer isgrown in potentiodynamic regime using high upper potential. Theconductive polymer can be further conditioned by the use of lowpotential eg 0V (vs Ag/AgCl) or by using a current step of 0 A for aperiod of 1-300 s.

After the electrochemical synthesis the polymer-coated sensors arepreferably washed with deionized water until monomer and sodiumdodecylsulphate are not traceable.

After the washing step the unbound water must be removed from thepolymer film. This may be done in several ways. The simplest way is toheat the sensors in an incubator for at least 8 hours. The temperaturecan be varied depending on the thickness of polymer film within therange 25-50° C., preferably 30-40° C. This range is very importantbecause on the one hand the unbound water cannot be completely removedat temperature lower than 25° C., on the other hand a high temperature(more than 50° C.) can damage the polymer film. Another possibility forremoving water is lyophilization.

The washing and drying steps described above may also be used to treatpolymer-coated electrodes prepared by the prior art methods ofelectrochemical polymerisation (see WO 96/02001, WO 00/11473 andreferences described therein). This treatment provides particularadvantages in relation to long-term storage of the electrodes, asdescribed above.

The main application for the polymer-coated electrodes obtained by themethods described above is production of highly sensitive potentiometricbiosensors e.g. chemical-, enzyme- or immunosensors. Any biologicalreceptor(s) can be immobilised onto a sensor using well known techniquesfor solid phase coating. Any form of redox, pH-changing or acidificationassay can be performed using these sensors including cell analysis.

In order to evaluate and control the redox sensitivity of the sensorsthe authors of the present invention propose to use a model assay basedon the use of polymer-based sensors coated with streptavidin, whichreact with biotinylated horseradish peroxidase. It has been mentionedabove that the use of commonly applied electrochemical procedures is notsuitable for testing the properties of the sensors as it is not possibleto distinguish the differences in properties at the required level. Animmunoassay helps to evaluate the redox properties and accordinglyanalytical sensitivity of the sensors prepared using differentpolymerisation regimes.

The techniques of coating the polypyrrole layer with streptavidin aredescribed in detail in the inventors' International application WO00/11473. The streptavidin concentration in the coating solution may bevaried from 2-100 μg/ml depending on the method of coating. In addition,in the present invention a protective sucrose layer is applied on thesensors coated with streptavidin followed by drying of the sensors.

The coating with streptavidin and application of protective layer can becombined together. In the latter case a drop (0.1-10 μl) of theprotective solution containing streptavidin is placed onto the sensorand dried. Other methods, e.g. screen-printing may be used.

The procedures for incubation with an analyte are described in WO00/11473. A wide range of aqueous buffers with different pH can beemployed for HRP solution preparation. The concentration of biotinylatedHRP is varied within range 0-100 ng/ml. The typical set ofconcentrations is: 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0,50.0, 100.0 ng/ml. The incubation time may be 2-60 min.

The potentiometric measuring procedure, as well as a calculation of ananalytical result, is described in detail in WO 00/11473.

It is well known that HRP requires the use of an H-donor to speed up theenzymatic reaction. However, in WO 00/11473 only H-donors which arecommonly used in routine immunoassays with optical detection werementioned. All these H-donors change their colour as a result of theenzyme reaction. This colour-change is not required for thepotentiometric measurement. In the latter case only the magnitude ofchange in redox state of the sensing element, e.g. polymer film as aresult of interaction with HRP is important. The authors of the presentinvention have found that a number of colourless substances can serve asH-donors for HRP and provide sufficient change in redox state to performpotentiometric measurement. Suitable H donors are those which give ahigh magnitude change in redox state as a result of the interaction withhorseradish peroxidase. Most preferably the H-donor will be an H-donorproviding a sensor potentiometric response of at least 10 mV as aconsequence of interaction with horseradish peroxidase under the definedconditions of the model assay. Examples of suitable colourless H-donorsare coniferol, guaiacol, MBTH. The concentration of H-donors used inpotentiometric measurement, depending on the particular H-donor, may bevaried within the range 0.1-100 mM. It is possible to extend or shiftthe measuring range for particular analyte by changing just the H-donorand/or its concentration in the substrate system.

In WO 00/11473 hydrogen peroxide served as the HRP substrate. Being astrong oxidising agent hydrogen peroxide may affect measuring results byinterfering with the polymer layer or underlying electrode. In thepresent invention it may be replaced with a substrate which is anorganic or non-organic peroxide. Suitable substrates includemethylhydroperoxide, ethylhydroperoxide or p-nitroperoxybenzoic acid andsodium perborate. The concentration of the substrate varies depending onthe nature of substrate within range of 0.0005-0.1%. In the case ofsodium perborate the preferred concentration is 0.03%.

Obviously the utility of these H-donors and peroxide substrates is notlimited to the model assay system for evaluation of redox sensitivityand quality control described herein. Indeed these H-donors andsubstrates may also be used for potentiometric analysis of variousanalytes, for example using methods analogous to the sandwich andcompetitive potentiometric analysis methods described in WO 00/11473.

These potentiometric analysis methods are analogous to the model assaysystem except that the surface of the sensor is modified with abiomolecule having specific binding affinity for the analyte of interestrather than streptavidin (NB the analyte-specific binding molecule maybe attached to the sensor via a biotin/streptavidin interaction withstreptavidin adsorbed to the sensor or immobilised in the polymercoating, as described in WO 00/11473). The features of potentiometricassays based on the use of an enzyme label (e.g. peroxidase) will beunderstood with reference to WO 00/11473. Enzymatic labels other thanthe HRP enzyme can be used (e.g. other peroxidases, glucose oxidase orcatalase) as any enzymatic process involves the electron transfer, whichin its turn change the potential of the sensor.

Typical “sandwich” potentiometric methods of electrochemical detectionusing an enzyme label (such as the methods described in WO 00/11473)comprise the steps of:

(a) providing a potentiometric sensor having an electroconductivepolymer coating, the coating having immobilized therein or adsorbedthereto receptors which are capable of binding to the desired analyte tobe detected in the sample;

(b) contacting the sensor with a test solution comprising the sample sothat the said analyte binds to said immobilized or adsorbed receptors;

(c) contacting the sensor with a solution comprising secondary receptorscapable of binding to said analyte at a site spatially distinct from thesite of binding to immobilized or adsorbed receptors, said secondaryreceptors being conjugated with at least one enzyme;

(d) monitoring the electric potential difference between the sensor anda reference electrode when both are immersed in a basic electrolytesolution;

(e) transferring the sensor and reference electrode to an enhancerelectrolyte solution having identical composition to the basicelectrolyte solution except that it additionally contains substrate forthe enzyme(s) and monitoring the electric potential difference betweenthe sensor and reference electrodes when immersed in the enhancerelectrolyte solution;

(f) calculating the difference between the electric potential differencemeasurements obtained in steps (d) and (e) and relating the resultobtained to the concentration of analyte in the sample.

Whereas, typical “competitive” potentiometric methods of electrochemicaldetection using an enzyme label (such as the methods described in WO00/11473) comprise the steps of:

(a) providing a potentiometric sensor having an electroconductivepolymer coating, the coating having immobilized therein or adsorbedthereto receptors which are capable of binding to the desired analyte tobe detected in the sample;

(b) contacting the sensor with a test solution comprising the sample sothat the said desired analyte binds to said immobilized or adsorbedreceptors;

(c) contacting the sensor with a solution comprising competing moleculescapable of binding to said immobilized or adsorbed receptors, saidcompeting molecules being conjugated with at least one enzyme;

(d) monitoring the electric potential difference between the sensor anda reference electrode when both are immersed in a basic electrolytesolution;

(e) transferring the sensor and reference electrode to an enhancerelectrolyte solution having identical composition to the basicelectrolyte solution except that it additionally contains substrate forthe enzyme(s) and monitoring the electric potential difference betweenthe sensor and reference electrodes when immersed in the enhancerelectrolyte solution;

(f) calculating the difference between the electric potential differencemeasurements obtained in steps (d) and (e) and relating the resultobtained to the concentration of analyte in the sample.

The invention provides methods having all the features of the typicalassays listed above, characterised in that (i) a peroxidase enzyme labelis used, optionally in conjunction with further enzyme labels selectedfrom peroxidases (e.g. horseradish peroxidase), glucose oxidase andcatalase, and (ii) the basic and enhancer electrolyte solutionscomprises an H-donor exhibiting a high magnitude of change in its redoxstate as a result on interaction with the peroxidase, thereby providinga high potentiometric sensor response.

Preferably the H-donor will provide a sensor potentiometric response ofat least 10 mV for an analyte concentration interest as a consequence ofinteraction with the peroxidase.

Most preferred H-donors include, but are not limited to, coniferol,guaiacol and MBTH.

Assays may also be performed using glucose oxidase or catalase as theenzyme label without peroxidase, but in this case it is not necessary toadd an H-donor to the electrolyte.

The invention further provides methods having all the features of thetypical assays listed above, characterised in that (i) the enzyme is aperoxidase (e.g. horseradish peroxidase, and (ii) the enzyme substrateis sodium perborate, hydrogen peroxide or an organic peroxide. In thesemethods the basic and enhancer electrolyte solutions comprise anH-donor, but the precise nature of the H-donor is not limited.

The possibility to use alternative H-donors (with a peroxidase enzymelabel) and different combinations of enzymes (e.g. combinations ofperoxidases, catalase, glucose oxidase, etc.) adds additionalflexibility for the whole system, expanding the range of possibleapplications for the present invention.

The invention will be further understood with reference to thefollowing, non-limiting, experimental examples:

EXAMPLES

All reagents were purchased from Sigma if not otherwise stated. Pyrrolepurchased from Merck was purified by distillation and stored in aliquotsat −20° C.

Example 1

This first example demonstrates the relationship between theconcentration of pyrrole, in the solution for the electrochemicalpolymerisation, and analytical sensitivity of the polypyrrole-basedpotentiometric sensors.

The working electrodes were custom-made planar electrodes comprising PET(polyethyleneterephthalate) support (˜125 μm) with the electro-depositedcopper (˜17 μm) coated with electrochemically-plated gold (˜30 μm). Theworking area was approximately 1.0 sq mm. Aqueous solutions forelectrochemical polymerisation comprised 0.0002M SDS (sodiumdodecylsulphate) serving as a supporting electrolyte and the followingpyrrole concentrations: 0.55M, 0.3M, 0.15M, 0.05M, 0.01M, 0.005M,0.0025M. One of solutions was placed in the cell for electrochemicalpolymerisation comprising the auxiliary platinum electrode and thereference electrode (BAS). Eight electrodes combined in one block havingone electrical contact were placed in the cell, the working areaimmersed in the solution. In order to provide uniform current densityall electrodes were placed in parallel to the auxiliary electrode. Inorder to minimise ohmic drop the reference electrode was located at thenearest possible distance from the working electrodes. Theelectrochemical polymerisation was carried out using μAutolab IIpotentiostat-galvanostat (EcoChemie), by applying cycling voltage within−0.2-+1.7 V four times with the scan rate 0.05 V/sec.

After polymerisation, the sensors were placed in a reservoir containingdeionized water, and the water was replaced systematically with fresh(every 15 min for 3 hours). After washing, the sensors were placed in anincubator at 37° C. for 24 hours.

Redox sensitivity was tested using a model assay (polypyrrole-basedsensors coated with streptavidin react with biotinylated horseradishperoxidase).

Dried sensors were placed in streptavidin solution (40 μg/mlstreptavidin in potassium phosphate buffer (0.05M, pH 8.0) at +4° C. for24 hours. After adsorption the sensors were placed in 10% aqueoussucrose solution for 1-2 min and then dried. After this step the sensorscan be foiled and kept for extended period of time. The estimated periodis at least 12 months.

In this example the protective sucrose film was removed by washing withthe reaction buffer (0.1M potassium phosphate buffer, pH 7.8) before thenext stage of the analysis. However, it is not essential for the sucroselayer to be removed at this stage. The sucrose-coated sensor can bebrought into contact with solution containing the analyte (in this casebiotinylated HRP). Sucrose is highly soluble and will dissolve veryquickly. Moreover the presence of sucrose in the reaction vessel doesnot affect the assay itself. In the present example, after washing outthe protective sucrose film the streptavidin-coated sensors wereincubated with various concentrations of biotinylated HRP in thereaction buffer, washed and left in it until the potentiometricmeasurement.

The method of potentiometric measurement, described in detail in WO00/11473, combines two steps. Typically, the first potentiometricmeasurement is taken (vs Ag/AgCl reference electrode) in the firstelectrolyte solution (additionally called the Basic Solution) containingan H-donor. The second potentiometric measurement is taken (vs Ag/AgClreference electrode) in the second electrolyte solution (additionallycalled the Enhancer Solution), which has the same chemical compositionas the Basic Solution, but with the addition of the enzyme substrate.The difference between two potentials related to the concentration of ananalyte in a sample is measured in millivolts. The presence of H-donorin the Basic Solution is desirable in order to eliminate thecontribution of H-donor itself to the final result.

The measurements were taken using a measuring device comprising themeasuring cell (constructed in these laboratories). The sensor wasplaced in the measuring cell, the Basic Solution was pumped in, and thenreplaced with the Enhancer solution according to set parameters. Theresult was calculated by custom-designed software.

In this example the Basic Solution was 100 mM OPD (o-phenilenediamine)in 0.05M sodium citrate buffer, pH 5.0. Sodium perborate (0.03%) wasused as a substrate for HRP in the Enhancer Solution. The firstpotentiometric measurement was taken at 20 sec; the secondpotentiometric measurement was taken at 60 sec.

The relationship between the potentiometric response and theconcentration of HRP for the sensors with the polypyrrole films, grownfrom the solutions with different concentrations of pyrrole, is shown inFIG. 1 a.

The response of polypyrrole-based sensor is strongly dependent on theconcentration of the monomer in the solution for electrochemicalpolymerisation. The relationship between the signal and theconcentration of pyrrole in the polymerisation solution for particularHRP concentration is shown on FIG. 1 b (0.1 ng/ml HRP), FIG. 1 c (1.0ng/ml HRP) and FIG. 1 d (10 ng/ml HRP).

The signal typically increases with a decrease of the concentration ofpyrrole in polymerisation solution. The ultimate increase in signal isobserved for the sensors produced from the solutions with 0.0025M-0.015Mof pyrrole (0.005M for 10 ng/ml, FIG. 1 d).

As mentioned above this effect is observed within the range of pyrroleconcentrations, which has not been considered by other researchers forproduction of highly sensitive polymer-based sensors.

This example demonstrated that the concentration of a monomer in thesolution for electrochemical polymerisation is one of the criticalfactors for production of highly sensitive potentiometric sensors.

Example 2

This example demonstrates the relationship between the concentration ofsupporting electrolyte in the solution for the electrochemicalpolymerisation and analytical sensitivity of the polypyrrole-basedpotentiometric sensors.

The potentiometric sensors were prepared as in Example 1, with thedifference, that the monomer concentration in the aqueous solutions forelectrochemical polymerisation was fixed (0.005M). SDS (supportingelectrolyte) was used in following concentrations: 0.0001M, 0.00015M,0.0002M, 0.0004M, 0.001M, 0.002M. The sensors were treated after thepolymerisation and the analytical sensitivity was tested accordingly asin Example 1 using biotinylated HRP concentrations within the range 0-10ng/ml.

The relationship between the potentiometric response and theconcentration of biotinylated HRP for the sensors with the polypyrrolefilms grown from the solutions with different concentrations of SDS isshown on FIG. 2 a. The response of polypyrrole-based sensor is stronglydependent on the concentration of the supporting electrolyte in thesolution for electrochemical polymerisation.

The relationship between the signal and the concentration of SDS in thepolymerisation solution for particular biotinylated HRP concentration isshown on FIG. 2 b (0.1 ng/ml HRP), FIG. 2 c (1.0 ng/ml biotinylated HRP)and FIG. 2 d (10 ng/ml biotinylated HRP). The curves have the peaks at0.0002M SDS, there is a drop at 0.00015M followed by sharp increase insensor response at 0.0001M for biotinylated HRP concentrations ≦1.0ng/ml.

The relationship between the SDS concentration in the polymerisationsolution and analytical sensitivity of the potentiometric sensors iscomplex. On the one hand SDS serves as a dopant ion providing certainelectrochemical properties to the polymer films, and the changes in SDSconcentration result in changes in analytical sensitivity of the sensors(see FIG. 2, a,b,c,d). On the other hand SDS serves as a supportingelectrolyte providing certain conductivity to the polymerisationsolution and the certain current density, which defines the thickness ofpolymer films. The polymer films grown from the polymerisation solutionswith higher SDS concentration are thicker.

The polymer films formed from the solutions with the lowest SDSconcentration (0.0001 M) are patchy. The potentiometric response ispartly derived from the polymer as a consequence of the enzyme reactionand partly by exposed gold appeared on the surface as a consequence ofsubstrate presence. The contribution of response of exposed gold isclearly seen at 0 ng/ml biotinylated HRP.

Both examples prove that the concentrations of the monomer andsupporting electrolyte and their ratio are critical factors responsiblefor analytical sensitivity of polymer-based potentiometric sensors. Theexamples also demonstrate the possibility to tailor the analyticalsensitivity and the measuring range by changing the parameters ofelectrochemical synthesis.

Example 3

This example demonstrates the relationship between parameters of thepolymerisation process and analytical sensitivity of polymer-basedpotentiometric sensors.

40 electrodes were combined in one block having one electrical contact.The electrodes were positioned on the perimeter of a round cell. AnAg/AgCl reference electrode was positioned in the centre of the cell.The auxiliary electrode was platinum gauze fixed to the bottom of thecell equidistant from each of the 40 working electrodes. The circledisposition was chosen in order to provide a uniform current density forall working electrodes. The polymerisation solution with theconcentrations of pyrrole (0.005M) and SDS (0.0002M) found optimal inprevious experiments (see examples 1 and 2) was used.

The electrochemical polymerisation was carried out using μAutolab IIpotentiostat-galvanostat (EcoChemie), by applying cycling voltage fourtimes with the scan rate 0.05 V/sec. The bottom boundary potential was−0.2 V (the same as in Example 1). The upper boundary potential waschanged within 1.4-2.0 V. The sensors were treated after thepolymerisation as described in previous examples and the analyticalsensitivity was tested accordingly as in Example 1 using biotinylatedHRP concentrations within the range 0-10 ng/ml.

According to the results the analytical response of the sensor and theshape of the curve (signal—HRP concentration) are influenced by upperboundary potential (FIG. 3).

The upper boundary potential is another critical factor forpotentiometric sensitivity of the potentiometric sensor. It isresponsible for redox state of the polymer film and its thickness, whichdepends on the quantity of electricity passed per surface area unit.

The example proves one of the main statements of the present inventionthat the concentrations of the monomer(s) and the supportingelectrolyte(s), the ratio between them, the range of applied cyclicvoltage synergistically influence analytical sensitivity ofpolypyrrole-based sensors.

It should be mentioned that the sensors produced by the method describedabove have higher sensitivity than known potentiometric sensors and canbe used for potentiometric analysis of a wide range of analytes.

Example 4

This example demonstrates the influence of the quantity of electricitypassed through working electrodes in galvanostatic regime ofelectrochemical synthesis on their analytical sensitivity.

40 electrodes were combined in one block having one electrical contact.The electrodes were positioned on the perimeter of a round cell. AnAg/AgCl reference electrode was positioned in the centre of the cell.The auxiliary electrode was platinum wire fixed to the bottom of thecell equidistant from each of the 40 working electrodes. Thepolymerisation solution with the concentrations of pyrrole (0.005M) andSDS (0.0002M) found optimal in previous experiments (see examples 1 and2) was used.

The electrochemical polymerisation was carried out using μAutolab IIpotentiostat-galvanostat (EcoChemie), by applying three successivedifferent current densities between electrodes, to be coated, andauxiliary electrode. The current density used was 0.1 mA/cm², 0.15mA/cm² and 0 mA/cm² for the first, second and third levels respectively.The duration of the first current density was varied within 300-900 s.The duration of the second and last density levels were kept constant(23 and 5 s respectively). Accordingly, the resulting quantity ofelectricity was 33.5-93.5 mC/cm².

The resulting sensors were treated after the polymerisation as describedin previous examples and their analytical sensitivity was testedaccordingly as in Example 1 using biotinylated HRP concentrations withinthe range 0-10 ng/ml.

The response of the sensor and the shape of the curve “signal—HRPconcentration” are dependent on the quantity of electricity passedthrough electrodes during polymerisation process (FIG. 4).

This example proves one of the main statements of the present inventionthat the concentrations of the monomer(s) and the supportingelectrolyte(s), the ratio between them, the quantity of electricitypassed through electrodes during polymerisation synergisticallyinfluence analytical sensitivity of polypyrrole-based sensors.

Example 5

This example demonstrates the influence of the application of more thanone polymerisation regime on the analytical sensitivity of the sensors.Two polymerisation protocols were used: conventional galvanostatic andcombined galvanostatic-potentiodynamic procedures. The total amount ofthe electricity and, consequently, the thickness of the polymer films inboth cases were the same.

Galvanostatic and potentiodynamic procedures are carried outsequentially using the polymerisation solution concentrations andpolymerisation cell format previously described in example 4. Theelectrochemical polymerisation was carried out using μAutolab IIpotentiostat-galvanostat (EcoChemie), galvanostatic current density was0.1 mA/cm² for 150 s (15 mC/cm²) followed by a single cyclic voltagescan with the scan rate 0.05 V/sec and a step potential of 2.44 mV. Thelower boundary potential is −0.2 V. The upper boundary potential is 1.90V (Procedure 1). Galvanostatic procedure was carried out with currentdensity 0.1 mA/cm² for 300 s (Procedure 2), which gave approximately thesame amount of electricity as in Procedure 1 (15 mC/cm²). Otherparameters were the same as for Procedure 1.

The resulting sensors were treated after the polymerisation as describedin previous examples and their analytical sensitivity was testedaccordingly as in Example 1 using two concentrations of biotinylated HRP(0 and 0.1 ng/ml). The results are in the table below.

Signal, mV Applied 0 ng/ml, 0.1 ng/ml, procedure [HRP-biotin][HRP-biotin] Procedure 1 14 51 Procedure 2 11 23

This example demonstrates the influence of application of twopolymerisation regimes. The potentiodynamic step can be ‘imitated’ byusing more than one level of current in galvanosatic procedure (seeprevious and next examples).

The application of two or more regimes allows more strict control of theredox properties of the polymer film and consequently to tailor theanalytical sensitivity of the sensors.

Example 6

This example demonstrates that by varying the parameters for thegalvanostatic regime the electrochemical polymer deposition can betailored to suit the requirements of a particular assay. In FIG. 5“Regime 1” produces sensors with high “sensitivity” (0 to 0.1 ng/mlresponse) but low “dynamic” range (0 to 10 ng/ml response) Sensorsproduced using “Regime 3” have a much larger dynamic range but lowersensitivity. The electrochemical polymerisation was carried out using aμAutolab II (EcoChemie) computer controlled electrochemical measurementsystem. Forty electrodes of a circular design (diameter=1.5 mm) wereplaced in a linear cell parallel to a platinum auxiliary electrode. Thecell was filled with the an aqueous solution containing pyrrole (7.5 mM)and SDS (0.17 mM). Polymer was deposited onto the electrodes using thegalvanostatic regime in which a series of current steps are performedgiving a total charge passed of between 13 to 24 mC/cm². After theelectrochemcial polymer deposition had ended, the sensors were treatedas described in previous examples and their analytical sensitivitydetermined using biotinylated HRP concentrations in the range 0 to 10ng/ml.

Example 7

This example demonstrates that different shaped electrode designsproduce sensors that respond slightly differently to the same levels ofbiotinylated HRP. In FIG. 6 a circular design (diameter=1.5 mm²) iscompared with a linear design (length=1 mm, width=0.25 mm). The circularelectrode design produces a sensor which has a lower dynamic range andlower sensitivity than the linear design.

Both sensors were produced using the galvanostatic regime, withconditions as described in previous examples. The total current passedduring polymer deposition was 24 mC/cm² for the circular electrode and210 mC/cm² for the linear electrode type.

REFERENCES

The contents of the following documents are to be considered asincorporated into the present application by reference.

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The invention claimed is:
 1. A method for producing highly sensitivepotentiometric sensors by coating of electrically conductive electrodeswith an electroconductive polymer, which method comprises the steps of:(a) preparing an aqueous solution for electrochemical polymerisationcomprising monomeric units of the electroconductive polymer at aconcentration in the range of 0.002-0.05M; and a supporting electrolyte,which also serves as a doping agent, at a concentration in the range of0.0001-0.005M; (b) assembling an electrochemical polymerisation cellcomprising the solution for electrochemical polymerisation, an auxiliaryelectrode, one or more working electrodes to be coated withelectroconductive polymer, and optionally a reference electrode; and (c)coating the working electrode(s) with a polymer film by theelectrochemical synthesis of polymer from the electrochemicalpolymerisation solution using at least one of the followingelectrochemical regimes: (i) applying a cyclic voltage in the range−0.2-+2.0 V vs Ag/AgCl reference electrode between the workingelectrode(s) to be coated and the auxiliary electrode; (ii) applying aconstant current in the single or multiple current steps with givencurrent density in a range 0.01-1 mA/cm² between working electrode(s) tobe coated and auxiliary electrode for defined period of time such thatfinal quantity of electricity passed through working electrode(s) willlie in a range 10-250 mC/cm²; (iii) applying a constant potential in asingle or multiple potential steps at the range 0-3 V between workingelectrode(s) to be coated and a reference electrode for defined periodof time such that final quantity of electricity passed through theworking electrode(s) will lie in a range 10-250 mC/cm².
 2. A methodaccording to claim 1 for producing highly sensitive potentiometricsensors by coating of electrically conductive electrodes with anelectroconductive polymer, wherein in step (c) two or more current stepsare applied in a galvanostatic regime.
 3. A method according to claim 1for producing highly sensitive potentiometric sensors by coating ofelectrically conductive electrodes with an electroconductive polymer,wherein in step (c) two or more potential steps are applied in apotentiostatic regime.
 4. A method, according to claim 1 for producinghighly sensitive potentiometric sensors by coating of electricallyconductive electrodes with an electroconductive polymer, wherein in step(c) two or more polymerisation regimes, are applied.
 5. A method,according to claim 4 for producing highly sensitive potentiometricsensors by coating of electrically conductive electrodes with anelectroconductive polymer, wherein in step (c) the two or morepolymerisation regimes are selected from the group consisting ofgalvanostatic, potentiodynamic, and potentiostatic regimes, are applied.6. A method according to claim 1 wherein in regime (i) the cyclicelectric potential is applied for 1-15 cycles.
 7. A method according toclaim 1 wherein in regime (ii) the number of applied current steps is1-5.
 8. A method according to claim 1 wherein in step (c) theelectrochemical regimes (i), (ii), (iii) and (iv) are performedsequentially or in any combination to coat the electrode(s) with apolymer film by the electrochemical synthesis of polymer from theelectrochemical polymerisation solution.
 9. A method according to claim8 wherein step (c) comprises performing electrochemical regimes (i) and(ii) or (ii) and (iii) sequentially.
 10. A method according to claim 1wherein the ratio between concentrations of monomeric units of theelectroconductive polymer and supporting electrolyte in theelectrochemical polymerisation solution is in the range 2:1 to
 30. 11. Amethod according to claim 10 wherein the ratio between concentrations ofmonomeric units of the electroconductive polymer and supportingelectrolyte in the electrochemical polymerisation solution isapproximately 25:1.
 12. A method according to claim 10 wherein the ratiobetween concentrations of monomeric units of the electroconductivepolymer and supporting electrolyte in the electrochemical polymerisationsolution is in the range 5:1 to 30:1.
 13. A method according to claim 1wherein the monomeric units of the electroconductive polymer arepyrrole, thiophene, furan or any mixture thereof.
 14. A method accordingto claim 1 wherein sodium dodecylsulphate is used as the supportingelectrolyte.
 15. A method according to claim 1 for use in production oftwo or more highly sensitive potentiometric sensors in a singlepolymerisation reaction, wherein in the cell for electrochemicalpolymerisation of step (b) two or more electrodes to be coated arecombined in one unit having one common electrical contact.
 16. A methodaccording to claim 15 wherein all electrodes to be coated inpotentiodynamic or potentiostatic regimes are positioned equidistantfrom the auxiliary electrode.
 17. A method according to claim 15 whereinall electrodes to be coated are positioned preferably equidistant fromthe reference electrode.
 18. A method according to claim 1, whichcomprises the additional steps of: (d) washing the electroconductivepolymer coated electrode(s) obtained in step (c) in deionized water; and(e) removing unbound water from the electroconductive polymer layer. 19.A method according to claim 18 wherein in step (d) the electroconductivepolymer coated electrode(s) are washed with deionized water until tracesof monomeric units of the electroconductive polymer and supportingelectrolyte are no longer detectable.
 20. A method according to claim 18wherein in step (e) the unbound water is removed from theelectroconductive polymer layer by heating the electrode(s) in anincubator for at least 8 hours.
 21. A method according to claim 20wherein the temperature of heating is within range 25-50° C., preferably30-40° C.
 22. A method according to claim 18 wherein in step (e) theunbound water is removed from the electroconductive polymer layer bylyophilization.